Skip to main content
. Author manuscript; available in PMC: 2016 Aug 1.
Published in final edited form as: Endocr Relat Cancer. 2015 Jul 10;22(5):713–724. doi: 10.1530/ERC-15-0287

Fig. 1. RAD1901 inhibits ESR1 activity in vitro and in vivo.

Fig. 1

A) Chemical structure of SERM RAD1901. B) SKBR3 cells were plated in phenol red free media supplemented with charcoal stripped FBS (CFS) 24 hours prior to transfection with an ERE-luciferase reporter together with ESR1 or ESR2 expression vectors. 24 hours after transfection, cells were treated with E2 (10 nM) together with RAD (10−10–10−6 M) for 24 hours prior to harvest and analysis of luciferase activity normalized to co-transfected β-galactosidase control. B) MCF7 cells were plated in phenol red free media supplemented with CFS 48 hours prior to treatment with 10−9 M E2 together with ICI 182,780 (ICI), RAD1901 (RAD), GW7604, or 4-hydroxytamoxifen (4OHT) (10−11–10−6 M) for 24 hours. mRNA levels of ESR1 target gene trefoil factor 1 (TFF1) were assessed using RT qPCR following RNA isolation. mRNA expression was normalized to the similarly detected 36B4 housekeeping gene, and expression levels are presented as fold change as compared to the vehicle-treated control. D) MCF7 cells were plated in phenol red free media supplemented with CFS 24 hours prior to treatment, and were treated with 10−9 M E2 as well as with the indicated ligands (10−11 – 10−6 M) on days 1, 4, and 6 of an 8 day proliferation assay. DNA content as assessed by fluorescence was measured as a surrogate for cell proliferation. The relative increase in DNA fluorescence was calculated by normalizing to baseline values detected in a duplicate plate of cells that was harvested on day 1 prior to the initial treatment. Data are representative of at least 3 independent experiments. E–F) MCF7 cell derived tumors were implanted into ovariectomized estrogen-treated nu/nu mice. When tumor volume reached ~0.1 cm3, animals (n = 9–10) were randomized to receive daily treatment with vehicle, tamoxifen (Tam, 20 mg/kg sc) or RAD (20 mg/kg sc). E) Mean tumor volume +/− SEM per day of treatment is presented. Significance (2-way ANOVA of matched values followed by Bonferroni comparison) as compared to the vehicle control is indicated (* p < 0.0001). F) Expression of ESR1 target genes in tumors was analyzed essentially as in (C).